Microspheres containing therapeutic agents and related methods of use

ABSTRACT

Microspheres, compositions including the microspheres, and methods of using the microspheres are disclosed herein. The microspheres can be substantially spherical and can include a copolymer of a monomer (such as an acrylic monomer) and a cyclodextrin or a derivative thereof. The microspheres can also include a therapeutic agent, such as a platinum-based drug.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application claims priority to U.S. Provisional PatentApplication No. 62/546,375, titled “MICROSPHERES CONTAINING THERAPEUTICAGENTS AND RELATED METHODS OF USE,” filed Aug. 16, 2017, which isincorporated herein by reference in its entirety.

TECHNICAL FIELD

The present disclosure relates to microspheres containing therapeuticagents, to compositions including the microspheres, and to methods forpreparing and using such microspheres and compositions. The microspheresand compositions can be used in the management or treatment of variousdiseases and disorders including cancer and other angiogenic-dependentdiseases by drug delivery and/or therapeutic embolization.

BRIEF DESCRIPTION OF THE DRAWINGS

The written disclosure herein describes illustrative embodiments thatare non-limiting and non-exhaustive. Reference is made to certain ofsuch illustrative embodiments that are depicted in the figures, inwhich:

FIG. 1 depicts an FTIR spectrum of microspheres prepared in accordancewith an embodiment of the present disclosure in comparison to FTIRspectra of reference microspheres.

FIG. 2 is an image of microspheres prepared in accordance with anembodiment of the present disclosure.

FIG. 3 is an image of microspheres prepared in accordance with anembodiment of the present disclosure.

FIG. 4 is an image of microspheres prepared in accordance with anembodiment of the present disclosure.

FIG. 5 is an image of microspheres prepared in accordance with anembodiment of the present disclosure.

FIG. 6 depicts Raman spectra of microspheres prepared in accordance withan embodiment of the present disclosure in comparison to a Ramanspectrum of reference microspheres.

FIG. 7 is an image of microspheres prepared in accordance with anembodiment of the present disclosure.

FIG. 8 depicts a Raman spectrum of microspheres prepared in accordancewith an embodiment of the present disclosure in comparison to a Ramanspectrum of reference microspheres.

FIG. 9 depicts an FTIR spectrum of microspheres prepared in accordancewith an embodiment of the present disclosure in comparison to an FTIRspectrum of reference microspheres.

DETAILED DESCRIPTION

The present disclosure relates to microspheres containing therapeuticagents, to compositions including the microspheres, and to methods forpreparing and using such microspheres and compositions. The microspheresand compositions can be used in the management or treatment of variousdiseases and disorders including cancer and other angiogenic-dependentdiseases by drug delivery and/or therapeutic embolization.

It will be readily understood that the embodiments, as generallydescribed herein, are exemplary. The following detailed description ofvarious embodiments is not intended to limit the scope of the presentdisclosure, but is merely representative of various embodiments. It willbe appreciated that various features are sometimes grouped together in asingle embodiment or description thereof for the purpose of streamliningthe disclosure. Many of these features may be used alone and/or incombination with one another. Moreover, the order of the steps oractions of the methods disclosed herein may be changed by those skilledin the art without departing from the scope of the present disclosure.In other words, unless a specific order of steps or actions is requiredfor proper operation of the embodiments, the order or use of specificsteps or actions may be modified.

Definitions

As used herein, the term “about” means within 20%, within 15%, within10%, within 5%, or within 1% or less of a given value or range. Further,all ranges include both endpoints.

As used herein, “administered,” “administering,” or “administration”refers to the act of injecting or otherwise physically delivering asubstance as it exists outside the body (e.g., a microsphere orcomposition) into a patient, such as by, but not limited to, pulmonary(e.g., inhalation), mucosal (e.g., intranasal), intradermal,intravenous, intramuscular delivery, and/or any other method of physicaldelivery described herein or known in the art. When a disease, orsymptoms thereof, is being managed or treated, administration of thetherapy (such as the microspheres disclosed herein) typically occursafter the onset of the disease or symptoms thereof. When a disease, orsymptoms thereof, is being prevented, administration of the therapy(such as the microspheres disclosed herein) typically occurs before theonset of the disease or symptoms thereof.

As used herein, “cell adhesion promoter” means any material that,because of its presence in or association with the microspheres,promotes or enhances the adhesiveness of cells to the surface of themicrospheres. These materials can include proteins that are associatedwith the surface of the microspheres through covalent bonds or in aninterpenetrated polymeric manner.

As used herein, the term “effective amount” refers to the amount of atherapy (e.g., a microsphere or composition disclosed herein) which issufficient to reduce and/or ameliorate the severity and/or duration of agiven disease and/or a symptom related thereto.

As used herein, “elastic” microspheres refer to microspheres thatcomprise polymers that have elastic properties.

As used herein, “hydrophilic” refers to microspheres or portions ofmicrospheres which may substantially bind with, absorb, mix easily with,and/or dissolve in water or aqueous solutions. This may result inswelling and/or the formation of reversible gels.

As used herein, “injectable” means capable of being administered,delivered, or carried into the body via syringe, catheters, needles, orother means for injecting or infusing the microspheres in a liquidmedium.

As used herein, the terms “manage,” “managing,” and “management” referto the beneficial effects that a subject derives from a therapy (e.g.,the microspheres disclosed herein), which does not result in a cure ofthe infection. In certain embodiments, a subject is administered one ormore therapies to “manage” a given disease or one or more symptomsrelated thereto, so as to prevent the progression or worsening of thedisease.

As used herein, “microspheres” means polymer or combinations of polymersmade into bodies of various sizes. The microspheres can be in any shape,although they are often substantially spherical. In certain embodiments,the microspheres are sterile, either alone or when in the form of apharmaceutical composition. The microspheres and compositions may besterilized by any method known in the art, for example, by irradiation,such as gamma or beta irradiation. The microspheres may comprise othermaterials as described and defined herein.

As used herein, “monomer composition” means any composition comprisingat least one monomer that may be polymerized to form a polymer orcopolymer. The monomer composition may optionally comprise othercomponents besides the at least one monomer. For example, the monomercomposition may comprise additional agents to aid in the polymerizationprocess, or it may comprise non-monomer compositions or components thatcan be incorporated or associated with the final polymer or copolymerafter polymerization. In the context of a polymer or copolymer, apolymer or copolymer comprises a monomer if the polymer or copolymer hasat least one of the monomer covalently bound to the polymer or copolymer(e.g., a polymerized monomer.)

The term “pharmaceutically acceptable” as used herein means beingapproved by a regulatory agency of the federal or a state government, orlisted in the U.S. Pharmacopeia, European Pharmacopeia, or othergenerally recognized pharmacopeia for use in animals, and moreparticularly in humans.

As used herein, “polymerize” or “polymerizing” means any action taken tocause one or more monomers to become covalently bound to form a polymeror copolymer. For example, a monomer composition or mixture may bepolymerized by adding an activating agent to the monomer composition ormixture to induce formation of a polymer or copolymer. In someembodiments, the activating agent comprisesN,N,N′,N′-tetramethylethylenediamine.

As used herein, the term “prevent” refers to the total or partialinhibition of a given disease; the total or partial inhibition of thedevelopment or onset of disease progression of given disease, or asymptom related thereto in a subject; or the total or partial inhibitionof the progression of a given disease or a symptom related thereto.

As used herein, “substantially spherical” generally means a shape thatis close to a perfect sphere, which is defined as a volume that presentsthe lowest external surface area. For example, “substantially spherical”can refer to microspheres wherein, when viewing any cross-section of themicrospheres, the difference between the major diameter (or maximumdiameter) and the minor diameter (or minimum diameter) is less than 20%,less than 15%, less than 10%, less than 5%, or less than 1% depending onthe embodiment used. The term “substantially spherical” can also referto a microsphere having a major diameter/minor diameter ratio of fromabout 1.0 to about 2.0, from about 1.0 to about 1.5, or from about 1.0to about 1.2.

As used herein, “swellable” microspheres refer to microspheres that arecapable of being enlarged in size, yet retain substantially the sameshape, upon certain conditions, such as contacting aqueous liquids orphysiological fluids.

As used herein, “therapeutic agent” refers to any substance thatprovides therapeutic effects to a disease or symptom related thereto. Incertain embodiments, a therapeutic agent refers to a substance thatprovides therapeutic effects to any of the processes ofangiogenesis-dependent diseases or biological or physiological responsesto the angiogenesis-dependent diseases. An example of a therapeuticagent is an anti-inflammation agent that prevents or reduces the effectof inflammations associated with angiogenesis-dependent diseases.

As used herein, the term “therapy” refers to any protocol, method,and/or agent that can be used in the management, treatment, and/oramelioration of a given disease, or a symptom related thereto. Incertain embodiments, the terms “therapies” and “therapy” refer to abiological therapy, supportive therapy, and/or other therapies known toone of skill in the art, such as medical personnel, useful in themanagement or treatment of a given disease, or symptom related thereto.

As used herein, “treat,” “treatment,” and “treating” refer to thereduction or amelioration of the progression, severity, and/or durationof a given disease resulting from the administration of one or moretherapies (including, but not limited to, the administration ofmicrospheres disclosed herein). In certain embodiments, the terms referto the reduction of pain associated with one or more diseases orconditions.

Microspheres

As further detailed herein, in some embodiments, the present disclosurerelates to microspheres suitable for drug delivery and/or therapeuticembolization. In an embodiment, the microspheres comprise a polymericmaterial. The polymeric material may comprise a copolymer comprising anacrylic monomer (and/or another monomer) and a cyclodextrin or aderivative thereof. The microspheres may also comprise a therapeuticagent. In an embodiment, the therapeutic agent is an anti-neoplasticdrug.

In some embodiments, the polymeric material is a biocompatible,polymeric material. Biocompatible, polymeric materials are non-toxic totissues and cells and generally do not cause inflammation. The polymericmaterial also comprises at least one polymer, copolymer, or mixturethereof. Further, as can be appreciated, when reciting that thepolymeric material comprises a particular monomer (e.g., an acrylicmonomer), it will generally be understood that the polymeric materialcomprises a polymerized form of such a monomer.

Various types of polymeric materials can be used, including naturaland/or synthetic polymeric materials. In some embodiments, the polymericmaterial comprises a copolymer comprising one or more acrylates,acrylamides, acrylics, vinyls, acetals, allyls, cellulosics,methacrylates, polyamides, polycarbonates, polyesters, polyimides,polyolefins, polyphosphates, polyurethanes, silicones, styrenes, and/orpolysaccharides, or derivatives and/or mixtures thereof. In particularembodiments, the polymeric material comprises a copolymer comprising atleast one of an acrylic monomer, acrylamide monomer, or vinyl monomer,or derivative thereof. Other polymerizable monomers can also be used.

In one embodiment, the polymeric material comprises a copolymercomprising an acrylamide monomer or a derivative thereof. For example,the polymeric material can comprise a copolymer comprising an acrylamidesuch as N-[tris(hydroxymethyl)methyl]acrylamide or a derivative thereof.Other acrylamides can also be used, including, but not limited to,methacrylamides, N,N′-methylenebis(acrylamide), diethyl aminoethylacrylamide, and triethyl aminoethyl acrylamide and derivatives thereof.In further embodiments, the polymeric material comprises a copolymercomprising a mixture of two or more different monomers, such as acrylicmonomers, acrylamide monomers, vinyl monomers, or derivatives and/ormixtures thereof. For example, the polymeric material can comprise acopolymer comprising a mixture of two or more of the herein-mentionedacrylamides or derivatives thereof, such as a mixture ofN-[tris(hydroxymethyl)methyl]acrylamide andN,N′-methylenebis(acrylamide) or derivatives thereof. In someembodiments, the polymeric material can further comprise an acrylate,such as sodium acrylate. For example, in some embodiments, the polymericmaterial comprises one or more (or two or more) acrylamides (e.g.,N-[tris(hydroxymethyl)methyl]acrylamide andN,N′-methylenebis(acrylamide)) and an acrylate (e.g., sodium acrylate).In some embodiments, the polymeric material lacks an acrylate monomer.Other monomer mixtures can also be used. For example, different monomershaving different properties can be polymerized to achieve a copolymerhaving one or more desired properties.

As further detailed below, in certain embodiments, the polymericmaterial further comprises a monomer having an alkyne functional group.In such embodiments, the monomer having an alkyne functional group canbe in addition to any of the above-mentioned monomers, such as acrylicmonomers, acrylamide monomers, vinyl monomers, or derivatives and/ormixtures thereof. As detailed below, the alkyne functional group can beuseful in bonding or coupling a cyclodextrin or derivative thereof to amicrosphere that has already been formed and/or polymerized. In some ofsuch embodiments, the monomer comprises an alkyne functional group and apolymerizable functional group. For instance, the monomer can comprise aterminal alkyne functional group at one end of the monomer. Monomershaving a cyclic alkyne group can also be used. The monomer can alsocomprise a polymerizable functional group, such as a terminalpolymerizable functional group at another end of the monomer. Exemplarypolymerizable functional groups include, but are not limited to,acrylics, vinyls, acrylamides, and acrylates. In some embodiments, thealkyne functional group is part of a propargyl group. The monomer canalso be water soluble.

Illustrative monomers having an alkyne functional group that can be usedinclude, but are not limited to, acrylamides, N-propargyl acrylamide,N-propargyl methacrylamide, acrylates, N-propargyl acrylate, N-propargylmethacrylate, But-3-ynyl prop-2-enoate,5-methyl-5-propargyloxycarbonyl-1,3-dioxane-2-one, and derivativesthereof. Other monomers having an alkyne functional group can also beused, including silicon-containing monomers such as trimethylsilylderivatives. For example, trimethylsilyl derivatives that include analkynyl group like 3-(trimethylsilyl) propargyl methacrylate can beused.

In one embodiment, the polymeric material comprises a monomer having analkyne functional group of formula I:

wherein X can be NR¹ or O, wherein R¹ is selected from H, CH₃, or C1-C6alkyl, or wherein R¹ is selected from H or CH₃,

wherein R is selected from H, CH₃, or C1-C6 alkyl, or wherein R isselected from H or CH₃, and

wherein n can be any integer from 1 to 20 or 2 to 20.

In another embodiment, the polymeric material comprises a monomer havingan alkyne functional group of formula II:

wherein X can be NR¹ or O, wherein R¹ is selected from H, CH₃, or C1-C6alkyl, or wherein R¹ is selected from H or CH₃,

wherein R is selected from H, CH₃, or C1-C6 alkyl, or wherein R isselected from H or CH₃, and

wherein n can be any integer from 1 to 20 or 2 to 20.

In yet another embodiment, the polymeric material comprisesalkyne-functionalized, cyclic monomers. For instance, the polymericmaterial can comprise alkyne-functionalized, cyclic carbonate monomerslike 5-methyl-5-propargyloxycarbonyl-1,3-dioxane-2-one, which can bedepicted as follows:

In still further embodiments, the polymeric material comprisesalkyne-functionalized monomers that include one or more polyetherchains. An illustrative monomer can be depicted as follows:

wherein R is a polymerizable group, such as an acrylamide or anacrylate.

Other types of polyether-like chains can also be used, including, butnot limited to polyethylene glycol (PEG), polypropylene glycol (PPG),polyoxymethylene (POM), and polytetrahydrofuran (PTHF).

In still further embodiments, the polymeric material comprisesalkyne-functionalized monomers that are part of cylcooctynes orderivatives thereof. An illustrative monomer can include a(1R,8S,9s)-Bicyclo[6.1.0]non-4-yn-9-yl group, which can be depicted asfollows:

wherein R is a polymerizable group, such as an acrylamide or anacrylate.

Another illustrative monomer can include a dibenzocyclooctyne group,which can be depicted as follows:

wherein R is a polymerizable group, such as an acrylamide or anacrylate. It will thus be appreciated that various types of monomershaving alkyne functional groups can be used in accordance with thepresent disclosure.

In some embodiments, the polymeric material comprises a mixture of atleast one acrylamide (e.g., N-[tris(hydroxymethyl)methyl]acrylamide) anda monomer having an alkyne functional group (e.g., N-propargylacrylamide). In some of such embodiments, the ratio, by weight, (ofacrylamide (e.g., N-[tris(hydroxymethyl)methyl]acrylamide):monomerhaving an alkyne functional group is between about 90:10 and about10:90; between about 80:20 and about 20:80; between about 80:20 andabout 30:70; between about 80:20 and about 40:60; between about 80:20and about 50:50; or between about 80:20 and about 60:40.

The copolymer also comprises at least one cyclodextrin or a derivativethereof. For example, in certain embodiments, the copolymer comprises anα (alpha)-cyclodextrin or a derivative thereof. Other cyclodextrins orcyclodextrin derivatives can also be used, including, but not limitedto, β (beta)-cyclodextrins and γ (gamma)-cyclodextrins and derivativesthereof. In further embodiments, the copolymer comprises a mixture oftwo or more cyclodextrins or derivatives thereof, such as two or more ofthe herein-mentioned cyclodextrins or derivatives thereof. As can beappreciated, α (alpha)-cyclodextrins can be used to describesix-membered sugar ring molecules, β (beta)-cyclodextrins can be used todescribe seven-membered sugar ring molecules, and γ(gamma)-cyclodextrins can be used to describe eight-membered sugar ringmolecules.

The amount of monomers (e.g., acrylamide monomers) and/or cyclodextrinsused can vary. In certain embodiments, for example, the copolymercomprises one or more monomers (e.g., acrylamide monomers, alkynefunctional monomers, non-cyclodextrin monomers, etc.) and one or morecyclodextrins or derivatives thereof. In some of such embodiments, thecopolymer comprises between about 10% and about 90% by weight of one ormore monomers, and between about 10% and about 90% by weight ofcyclodextrins (or derivatives thereof). In other embodiments, thecopolymer comprises between about 10% and about 80%, between about 20%and about 80%, between about 30% and about 80%, between about 40% andabout 80%, or between about 50% and about 80% by weight of one or moremonomers, and between about 20% and about 90%, between about 20% andabout 80%, between about 20% and about 70%, between about 20% and about60%, or between about 20% and about 50% by weight of cyclodextrins (orderivatives thereof).

In some embodiments, the cyclodextrin is modified or functionalized suchthat it can be polymerized and incorporated into the polymeric material.For example, the cyclodextrin can be modified or functionalized toinclude a group or moiety that can be polymerized with another monomer(e.g., such as an acrylic monomer or a monomer having an alkynefunctional group as described herein) to form a copolymer. In particularembodiments, the cyclodextrin is modified or functionalized to include agroup or moiety that can be polymerized with an acrylic monomer (and/oranother monomer) and incorporated into the backbone or backbone chain ofa copolymer. In some embodiments, the cyclodextrin is modified toinclude an olefin.

In one embodiment, the modified cyclodextrin is selected from the groupconsisting ofN-propenoyl-1-(6-deoxy-β-D-cyclodextrin-4-aminomethyl-1,2,3 triazole,N-propenoyl-1-(6-O-hexyl-β-D-cyclodextrin)-4-aminomethyl-1,2,3 triazole,N-propenoyl-1-(6-hexylamino-6-deoxy-β-D-cyclodextrin-4-aminomethyl-1,2,3triazole, and 6-monoazido-6-monodeoxy-β-cyclodextrin or mixturesthereof.

The cyclodextrin can be modified or functionalized in various ways, someof which are illustrated below. However, it will be appreciated that thereaction mechanisms detailed below are illustrative and not meant to beexhaustive or limiting in any way. In some embodiments, a tosylchloride, N-tosylimidazole, mesyl chloride, or other like compound(e.g., sulfonyls etc.) can be reacted with a cyclodextrin. In someembodiments, the tosyl chloride, N-tosylimidazole, mesyl chloride, orother like compound is added to the cyclodextrin at a molar ratio ofbetween 1:1 and 3:1.

In some embodiments, this step of modifying a cyclodextrin with a tosylchloride, N-tosylimidazole, mesyl chloride, or other like compound iscarried out in an aqueous or organic solvent. For example, in someembodiments, this step is carried out in a solvent that comprises orconsists of water, pyridine, or a combination thereof. In someembodiments, this step is carried out at a temperature of between 10° C.and 60° C., such as between 15° C. and 55° C. In some embodiments, thereaction is carried out for between one hour and 48 hours (e.g., between2 hours and 24 hours). In some embodiments, one or more ofN,N′-dicyclohexylcarbodiimide (DCC), 4-dimethylaminopyridine (DMAP), orNaOH serves as a reagent during this step.

The resulting tosylate groups, mesylate groups, or other like groups(other sulfonate groups, etc.) can then be displaced by an azide (orother like compound) and subjected to a click reaction, such as a clickreaction with an alkyne containing an acrylic group (or otherpolymerizable group) to form a modified or functionalized cyclodextrinas shown below:

wherein X can be NR¹ or O, wherein R¹ is selected from H or C1-C6 alkyl,and

wherein n can be any integer from 1 to 20 or 2 to 20.

In some embodiments, the addition of azide is carried out in a solventcomprising or consisting of dimethylformamide (DMF). In otherembodiments the addition of an azide is carried out in a solventcomprising or consisting of water. In some embodiments, the reaction iscarried out at a temperature of between 15° C. and 80° C., such asbetween 10° C. and 30° C. or between 70° C. and 90° C. The clickreaction of an alkyne with an azide can be carried out in any suitablesolvent (e.g., dimethyl sulfoxide (DMSO)) and can include one of morecatalysts, such as a Cu²⁺ catalyst (e.g., CuSO₄). The alkyne, may be anysuitable alkyne, such as N-(prop-2-yn-1-yl)acrylamide.

In another embodiment, the cyclodextrin can be modified orfunctionalized through use of a reduction reaction. For example, tosylchloride, mesyl chloride, or other like compound (e.g., sulfonyls etc.)can be reacted with a cyclodextrin. The resulting tosylate groups,mesylate groups, or other like groups (other sulfonate groups, etc.) canthen be displaced by an azide (or other like compound) and reduced to anamine (or other like group), which can be further reacted with acompound containing an acrylic group (or other polymerizable group),such as acryloyl chloride, to form a modified or functionalizedcyclodextrin as shown below:

wherein X can be OH or a halogen such as Cl.

In some embodiments, the reduction step is carried out in the presenceof palladium on carbon (Pd/C). In other embodiments, the reduction stepis carried out in the presence of a phosphine (e.g.,triphenylphosphine). In other words, in some embodiments, an azide maybe reduced to an amine via a Staudinger reduction.

In another embodiment, the cyclodextrin may be modified orfunctionalized via an amination reaction. For example, in someembodiments, a tosylated cyclodextrin is modified by the addition of aprimary amine. In some embodiments, this step is carried out in amicrowave oven. In some embodiments, this reaction is carried out in asolvent such as DMF.

wherein R is a moiety containing at least one carbon atom.

In another embodiment, the cyclodextrin can be modified orfunctionalized through direct substitution of one or more hydroxylgroups (e.g., primary hydroxyl groups) on the cyclodextrin. For example,one or more hydroxyl groups (e.g., primary hydroxyl groups) can bedirectly substituted with an amine, diamine, or other like compound,which can be further reacted with a compound containing an acrylic group(or other polymerizable group), such as acryloyl chloride, to form amodified or functionalized cyclodextrin as shown below:

wherein X can be OH or a halogen such as Cl, and

wherein n can be any integer from 1 to 20 or 2 to 20.

In another embodiment, one or more hydroxyl groups (e.g., primaryhydroxyl groups) can be directly substituted with an azane, ammonia,ammonium hydroxide, or other like compound, which can be further reactedwith a compound containing an acrylic group (or other polymerizablegroup), such as acryloyl chloride, to form a modified or functionalizedcyclodextrin as shown below:

wherein X can be OH or a halogen such as Cl.

In some embodiments, one or more chain extenders (or spacers) can alsobe used. For example, one or more chain extenders can be included toincrease the distance between the polymerized group and the cyclodextringroup of the functionalized cyclodextrin.

The cyclodextrin can be monofunctionalized, difunctionalized, orpolyfunctionalized as desired. For example, the functionalizationreactions, such as the above-mentioned functionalization reactions, canbe performed once, twice, or more times to achieve a cyclodextrin havinga desired number of functionalized groups. In some embodiments whereinthe cyclodextrin is modified as a difunctional or polyfunctionalcyclodextrin, the two or more polymerizable or functional sites can alsocross-link the copolymer. An illustrative difunctionalized cyclodextrinhaving two polymerizable groups can be depicted as follows:

wherein X can be O or NR¹, wherein R¹ is selected from H or C1-C6 alkyl,

wherein Z can be O or NR², wherein R² is selected from H or C1-C6 alkyl,and

wherein n can be any integer from 1 to 20 or 2 to 20.

An illustrative polyfunctional cyclodextrin is depicted below. In suchan example, each of the primary alcohol groups on the cyclodextrin hasbeen substituted (fully substituted). One or more of the substitutedgroups can then be further modified as desired. For example, apolymerizable group can be added as follows:

wherein X can be O or NR¹, wherein R¹ is selected from H of C1-C6 alkyl,

wherein Z can be O or NR², wherein R² is selected from H of C1-C6 alkyl,and

wherein n can be any integer from 1 to 20 or 2 to 20.

One or more protecting groups can also be used when modifying orfunctionalizing the cyclodextrin. For example, protecting groups ormethods can be used to selectively functionalize a cyclodextrin.Illustrative protecting groups or methods include, but are not limitedto, selective debenzylation methods. An illustrative selectivedebenzylation method that can be used to selectively functionalize acyclodextrin can be depicted as follows:

Further modification of the selected hydroxyl groups can then beperformed to selectively add polymerizable groups at the selected sites.

Other types of protecting groups and/or selective functionalizationmethods can also be used. For example, in one embodiment a cappingmoiety (or other protecting moiety) can be used. Various types ofcapping moieties and/or protecting moieties can be used. In someembodiments, a capping moiety or other protecting group can be used tobind the polymerizable group to a cyclodextrin as follows:

wherein x can be O, NR¹, or S, wherein R¹ is selected from H or C1-C6alkyl,

wherein X² can be O or NR², wherein R² is selected from H or C1-C6alkyl,

wherein n can be any integer from 1 to 20 or 2 to 20.

In other embodiments, a capping moiety or other protecting group can beused to protect or selectively bind the polymerizable group onto acertain site of the cyclodextrin. In such embodiments, the cappingmoiety or other protecting group can thereafter be removed (or decapped)from the cyclodextrin, depicted as follows:

wherein X² can be O or NR², wherein R² is selected from H or C1-C6alkyl,

wherein x can be O, NR¹, or S, wherein R¹ is selected from H or C1-C6alkyl,

wherein n can be any integer from 1 to 20 or 2 to 20.

As can be appreciated, a modified and/or functionalized cyclodextrin canthen be polymerized with one or more other monomer units, such as any ofthe herein-identified monomers. For example, the modified and/orfunctionalized cyclodextrin can be polymerized such that it forms asegment of the polymer backbone. An illustrative polymerization of afunctionalized cyclodextrin with other monomer units is illustrated asfollows:

In certain embodiments, the polymeric material is cross-linked. Forexample, in some embodiments, the modified and/or functionalizedcyclodextrin can function as a cross-linker. In other embodiments, thepolymeric material can be cross-linked with a cross-linking agent. Insome embodiments, the cross-linking agent comprises an aldehyde, suchas, for example, glutaraldehyde or formaldehyde. In other embodiments,the cross-linking agent comprises an acrylamide, such as, for example,N,N′-methylene-bis-acrylamide, N′,N′-diallylacrylamide, orglyoxal-bis-acrylamide. In further embodiments, the cross-linking agentcomprises gelatin. Other types of cross-linking agents can also be used.In yet other embodiments, the polymeric material is not cross-linked.

In particular embodiments, the degree of crosslinking (or the amount ofcross-linking agent) is between about 1% and about 30% by mol ofcross-linking agent based on the total amount of the one or moremonomers. In other embodiments, the degree of crosslinking (or theamount of cross-linking agent) is between about 5% and about 15% by molof cross-linking agent based on the total amount of the one or moremonomers.

The microspheres comprising cyclodextrin can be characterized in manyways. In some embodiments, the microspheres comprising cyclodextrin arecharacterized using fourier transform infrared spectroscopy (“FTIR”). Incertain embodiments, the microspheres comprising cyclodextrin exhibit aFTIR peak (performed on dry microspheres at room temperature (about 23°C.)) that is distinguishable from or different than the baseline (and/ornoise) at between about 1140-1160 cm⁻¹, or between about 1145-1155 cm⁻¹.No such peak is exhibited by microspheres that do not includecyclodextrin.

The microspheres can also comprise a therapeutic agent. For example, themicrospheres can be loaded with a therapeutic agent. In other words, thetherapeutic agent can be adsorbed, absorbed, or otherwise associatedwith or bound to the microspheres. In a particular embodiment, thetherapeutic agent associates with or otherwise interacts with acyclodextrin group or moiety of the polymeric material. For example, thecyclodextrin group or moiety can comprise a ring or ring-like structure.The ring or ring-like structure can be substantially toroidal in shape.In some embodiments, the therapeutic agent can be at least partiallyloaded or disposed within the substantially toroidal shape. Thetherapeutic agent can also be described as being bound to the inside orinner surface of the ring or ring-like structure or toroidal shape. Inother embodiments, the cyclodextrin group or moiety can be described ashaving a cavity disposed within the ring or ring-like structure. In suchembodiments, the therapeutic agent can be at least partially loaded ordisposed within the cavity of the ring structure.

In some embodiments, the therapeutic agent (e.g., drug) is loaded intodry microspheres at a ratio of from 0.05:1 to 10:1 by weight, from 0.1:1to 10:1 by weight, from 1:2 to 5:1 by weight, or from 1:2 to 2:1 byweight. For instance, in some embodiments, 10 to 100 mg of therapeuticagent is loaded into 50 to 200 mg of dried microspheres. In someembodiments, a solution comprising therapeutic agent is combined withmicrospheres at a volumetric ratio of 1:1 to 100:1, or from 1:5 to 1:50.For example, in some embodiments, 1 mL to 2 mL of microspheres is addedto 5 mL to 100 mL of a drug solution.

As further detailed herein, the therapeutic agent can be released fromthe microspheres when used in a therapeutic procedure. For example, insome embodiments, the cyclodextrin group or moiety degrades, ismetabolized, or otherwise breaks down during a therapeutic procedure.For example, the cyclodextrin group or moiety can degrade, bemetabolized, or break down in vivo when subjected to physiologicalconditions. In such embodiments, the therapeutic agent can be releasedfrom the microspheres as the cyclodextrin group or moiety degrades.

Various types of therapeutic agents can be used. For example, in someembodiments, the therapeutic agent comprises an anti-neoplastic drug,such as, for example, a chemotherapeutic drug. In particularembodiments, the therapeutic agent comprises a platinum-based drug orderivative thereof. For example, the therapeutic agent can comprise oneor more of cisplatin, carboplatin, oxaliplatin, oxiplatin, satraplatin,picoplatin, nedaplatin, triplatin, lipoplatin, spiroplatin, iproplatin,and derivatives and mixtures thereof. Other platinum-based drugs orderivatives thereof can also be used. In one embodiment, the therapeuticagent comprises at least one of cisplatin, carboplatin, oxaliplatin, andoxiplatin. Other types of therapeutic agents can also be used. Forexample, in some embodiments, the microspheres comprise one or moretaxols or derivatives thereof, such as one or more of docetaxel orpaclitaxel. In another embodiment, the microspheres comprise one or moreanthracyclines or derivatives thereof, such as one or more ofdoxorubicin, daunorubicin, epirubicin, idarubicin, valrubicin, andmitoxantrone. In another embodiment, the microspheres comprise one ormore camptothecins or derivatives thereof, such as one or more ofirinotecan, topotecan, exatecan, and lurtotecan. Mixtures of any of theherein-mentioned therapeutic agents can also be used. In furtherembodiments, the therapeutic agent comprises any therapeutic agentcapable of being loaded or bound within the ring or ring-like structureof a cyclodextrin group or moiety.

In certain embodiments, the microspheres are spherical or substantiallyspherical in shape. The diameter of the microspheres may vary. Forexample, in some embodiments, the microspheres have an average diameterof from about 10 μm to about 2,000 μm, from about 30 μm to about 1,500μm, from about 40 μm to about 1,200 μm, from about 40 μm to about 900μm, from about 40 μm to about 600 μm, or from about 40 μm to about 400μm.

The microspheres can also be substantially uniform in size. For example,the difference in diameter between individual microspheres can be fromabout 0 μm to about 250 μm, from about 0 μm to about 200 μm, from about0 μm to about 150 μm, or from about 0 μm to about 100 μm. In furtherembodiments, individual microspheres have differences in diameter of 200μm or less, 150 μm or less, 100 μm or less, about 50 μm or less, about25 μm or less, about 10 μm or less, or about 5 μm or less. In particularembodiments, the microspheres are in a population wherein greater than68% have a diameter of ±20% of the mean, ±10% of the mean, or ±5% of themean diameter. In one embodiment, the microspheres are in a populationwherein greater than 75% have a diameter of ±20% of the mean, ±10% ofthe mean, or ±5% of the mean diameter.

In some embodiments, the microspheres are substantially hydrophilic. Forexample, the microspheres can contain at least one hydrophilic polymeror copolymer. In some of such embodiments, the microspheres can alsoinclude one or more hydrophobic polymers or copolymers as long as theoverall characteristic of the microspheres are substantially hydrophilicrather than hydrophobic. In some embodiments, the hydrophilic polymer orcopolymer is a polymer or copolymer containing —OH and/or —NH₂ groups.In other embodiments, the hydrophilic polymer or copolymer containsionic groups.

In certain embodiments, the microspheres are swellable and/or waterswellable. For example, the microspheres can be swellable upon contactwith a pharmaceutically acceptable liquid, such as water, buffersolutions, saline, body liquids, physiological fluids, and aqueous saltsolutions. For example, in particular embodiments, the microspheres canswell such that they are enlarged to about 15 times their original size(i.e., diameter) or to about 3,375 times their original volume. In otherembodiments, the microspheres can swell such that they are enlarged toabout four times their original size (i.e., diameter) or 64 times involume upon contact with saline (e.g., 0.9% sodium chloride solution).In certain embodiments, the microspheres can swell such that they areenlarged to at least about 110%, at least about 115%, at least about120%, at least about 125%, at least about 130%, at least about 135%, atleast about 140%, at least about 145%, or at least about 150% theiroriginal diameter upon contact with water.

In some embodiments, swellable microspheres refer to microspheres thathave the ability to absorb water. For example, in certain embodiments,the water absorption rate of a swellable microsphere is at least about750 g/g. The degree of swelling can be controlled by controlling factorssuch as, for example, the solvents in which the microspheres aresuspended, and specific polymers or copolymers used to make themicrospheres. In certain embodiments, the degree of crosslinking can beadjusted, and in other embodiments, cross-linking is not adjusted or isnot present.

In some embodiments, the microspheres can also be flexible or elasticsuch that they can easily pass into and through injection devices andsmall catheters without being permanently altered. The microspheres canalso be resistant to the muscle contraction stress generated during andafter the implantation process. The microspheres can also becompressible.

The microspheres can also be substantially non-aggregating such thatthey do not dump together. Further, in some embodiments, themicrospheres do not substantially adhere to the wads of storagecontainers and/or implantation devices, such as catheters, syringes,needles, and the like.

In some embodiments, the microspheres are substantially insoluble inpharmaceutically acceptable liquids, such as water, buffer solutions,saline, body liquids, and physiological fluids. In particularembodiments, the microspheres are substantially water insoluble. Infurther embodiments, a first portion of the microsphere is soluble and asecond portion of the microsphere is insoluble in water and/or otherpharmaceutically acceptable liquids. For example, the cyclodextringroups or moieties can be soluble whereas the remaining portion of thecopolymer can be insoluble in water and/or other pharmaceuticallyacceptable liquids. In some of such embodiments, the microspheres canretain their substantially spherical shape after degradation of thecyclodextrin groups or moieties.

In certain embodiments, the microspheres are non-resorbable and/ornon-biodegradable. For example, in such embodiments, the microspheresare not capable of being eliminated by the immune or lymphatic system.In other embodiments, the microspheres are resorbable and/orbiodegradable. In yet other embodiments, a portion of the microsphere isnon-resorbable and/or non-biodegradable while a second portion of themicrosphere is resorbable and/or biodegradable. For example, thecyclodextrin groups on the copolymer of the microspheres can bedegradable and/or resorbable while the remainder of the copolymerremains non-resorbable. In some of such embodiments, the microspheresare non-resorbable and/or non-biodegradable after degradation of thecyclodextrin groups or moieties. In further embodiments, themicrospheres, including the cyclodextrin groups and the remainingcopolymer, are resorbable and/or biodegradable.

In some embodiments, the microspheres are configured for use an embolicagents. In some embodiments where a first portion of the microsphere isbiodegradable and/or resorbable (e.g., a cyclodextrin-containingportion) but a second portion of the microsphere is non-biodegradableand/or non-resorbable, the microspheres may retain their function asembolic agents even after the first portion of the microsphere has beendegraded. Stated differently, in some embodiments, the microspheres areconfigured to substantially prevent the flow of blood through a regionof the vasculature, even after a therapeutic agent (or a portionthereof) has been released from the microspheres due to degradation of acyclodextrin-containing portion of the microsphere. In some embodiments,the microspheres may completely prevent blood flow through a region ofthe vasculature. In other embodiments, the microspheres may decrease theflux of blood flow across a region of the vasculature by more than 80%,more than 90% and/or more than 95%. In some embodiments, the change influx resulting from partial degradation of the microspheres is less than5%. Stated differently, when administered to a patient, the microspheresmay decrease the flux of blood flow across a region of the vasculatureof the patient by 90% or more regardless of whether the biodegradableportion of the microsphere has been degraded.

In some embodiments, the microspheres further include or are used withmarking agents. Exemplary marking agents include, but are not limitedto, dyes, imaging agents, and contrast agents (e.g., ionic and non-ioniccontrast agents).

In certain embodiments, the microspheres further include or are coatedwith agents which promote cell adhesion (e.g., cell adhesion promoters).For example, various types of cell adhesion promoters can be used.Exemplary cell adhesion promoters that can be used include, but are notlimited to, collagen, gelatin, glucosaminoglycans, fibronectins,lectins, polycations (such as polylysine, chitosan, and the like), orany other natural or synthetic biological cell adhesion agent. Inspecific embodiments, living cells are attached to the microspheres,forming layers of cells therein or thereon that link with surroundingtissues and can enhance the long-term stability of the microspheres.

In some embodiments, the microspheres are sterile. The microspheres canalso be thermally stable which allows for easy, convenient sterilizationand room temperature, refrigerated, or frozen storage. The microspherescan also be stable in suspension, which allows the microspheres to beformulated and stored in suspension and injected with different liquids.For example, in some embodiments, the microspheres can be placed insuspension, and in particular, in the form of sterile and pyrogenic(pyrogen-free) injectable solutions, while avoiding the formation ofaggregates or adhesion to the walls of storage containers andimplantation devices, such as catheters, syringes, needles, and thelike.

Methods of Preparing the Microspheres

The present disclosure also relates to methods of preparing ormanufacturing the microspheres. For example, in some embodiments, themethod comprises polymerizing a monomer composition comprising a monomer(e.g., such as an acrylic monomer, an acrylamide monomer, and/or anothermonomer described herein) and a cyclodextrin or a derivative thereof(e.g., such as a cyclodextrin described herein). The monomer compositionmay be polymerized according to various polymerization methods,including, but not limited to, suspension polymerization methods anddrop-by-drop polymerization methods. In certain embodiments, themicrospheres can be prepared in accordance with the polymerizationmethods described in French Patent No. 2,378,808 or U.S. Pat. Nos.5,648,100, and 5,635,215, each of which is incorporated herein byreference.

In some embodiments, the microspheres are prepared by adding the monomercomposition to an oil or other organic phase, Exemplary oils include,but are not limited to, mineral oils, paraffin oils, silicon oils, etc.In particular embodiments, the oil is heated before adding the monomercomposition to the oil. For example, the oil can be heated to atemperature of from about 20° C. to about 100° C., or from about 30° C.to about 80° C. In certain embodiments, after adding the monomercomposition to the oil, the resulting suspension is stirred during whichthe monomer composition is allowed to polymerize and microspheres areformed. In particular embodiments, the speed at which the suspension isstirred will change the distribution of the diameters of themicrospheres that are formed. As can be appreciated, the monomercomposition or the organic phase (e.g., oil) can also include variousadditives and/or additional agents, including, but not limited to,cross-linking agents, activating agents (e.g.,N,N,N′,N′-tetramethylethylenediamine), surfactants, salts, buffers,etc., and mixtures thereof.

In some embodiments, one or more cyclodextrin derivatives that are usedto form the microspheres are configured for polymerization with one ormore other monomers. For example, in some embodiments, a cyclodextrinderivative may include an olefin group that is suitable forpolymerization with another olefin-containing monomer to form a backboneof a polymeric structure that forms the microsphere.

In other embodiments, microspheres are initially prepared from a polymerthat is formed from monomers that do not contain cyclodextrin. After themicrospheres (which lack cyclodextrin) have been formed, one or morecyclodextrin derivatives are then attached to the microspheres. In otherwords, in some embodiments, the initial formation of the microspheresdoes not involve a cyclodextrin-containing component. But once themicrospheres are formed, one or more cyclodextrin derivatives are thenattached to the microspheres.

For example, in some embodiments, microspheres are initially formed frommonomers that do not include a cyclodextrin group, such as any of theabove-identified non-cyclodextrin-containing polymers. The microspheresthat are initially formed from monomers that lack a cyclodextrin groupmay include one or more functional groups that are configured tofacilitate subsequent attachment of one or more cyclodextrin groups tothe microspheres. For example, in some embodiments, the microspheresinclude a functional group selected from the group consisting of analkyne, an amine, or a carboxylic acid/carboxylate.

In one embodiment, the microspheres include an alkyne functional groupthat reacts with an azide-containing cyclodextrin derivative (e.g., viaclick chemistry) to form a triazole adduct.

For example, the microsphere can be formed by polymerizing one or moremonomers having an alkyne functional group or a propargyl functionalgroup. In one such embodiment, for instance, a monomer having an alkynefunctional group (e.g., N-propargyl acrylamide) can be polymerized withone or more additional monomers, such as acrylamide monomers or any ofthe other above-identified non-cyclodextrin-containing monomers andformed into a microsphere. This microsphere can include an alkynefunctional group that can be reacted with an azide-containing or othercyclodextrin derivative as shown in the reaction scheme above.

In another embodiment, the microspheres include a terminal aminefunctional group that reacts with a cyclodextrin derivative (e.g., atosylated β-cyclodextrin), thereby attaching one of more cyclodextringroups to each microsphere.

In some embodiments, the terminal amine functional group is attached tothe microsphere via a spacer. The terminal amine then reacts with thederivatized cyclodextrin, thereby attaching one or more cyclodextringroups to each microsphere.

In some embodiments, the microspheres initially include a carboxylicacid or carboxylate functional group. The carboxylic acid/carboxylategroup may be reacted with a terminal amine of a cyclodextrin derivative,thereby attaching one or more cyclodextrin groups to each microsphere,

In some embodiments, the microspheres are washed after polymerizationand/or attachment to a cyclodextrin derivative. For example, themicrospheres can be washed with water or an aqueous solution (e.g., asalt solution). In further embodiments, the microspheres are separatedand/or sieved. For example, the microspheres can be sieved to obtain apopulation of microspheres having an average diameter within a desiredrange.

In some embodiments, the polymerized monomers (including thecyclodextrins) are dispersed throughout the body (or polymer body) ofthe microsphere. For example, the polymerized monomers (including thecyclodextrins) can be dispersed or distributed (e.g., uniformly orsubstantially uniformly) throughout the polymer matrix that forms themicrosphere. In other embodiments, cyclodextrin groups are positionedonly on the outer surfaces or outer regions of the microspheres, but arenot found in interior regions of the microsphere.

In certain embodiments, the microspheres are loaded with a therapeuticagent. For example, the microspheres can be contacted with a therapeuticagent prior to use in a therapeutic procedure. In some embodiments, themicrospheres are suspended in a liquid and a therapeutic agent is addedto the suspension. In other embodiments, the microspheres are added to asolution comprising the therapeutic agent.

Pharmaceutical Compositions

The present disclosure also relates to pharmaceutical compositionscomprising the microspheres disclosed herein. For example, in anembodiment, a pharmaceutical composition is disclosed that comprisesmicrospheres and a pharmaceutically acceptable liquid or otherbiocompatible carrier. The compositions can be in the form of asuspension, a hydrogel, or an emulsion. For example, the composition cancomprise a suspension of microspheres in a pharmaceutically acceptableliquid or other biocompatible carrier. In some embodiments, thecompositions are sterile.

In certain embodiments, the pharmaceutically acceptable liquid can be,without limitation, saline, a buffer-solution, water, an isotonicsolution, a biological fluid, or a mixture thereof. The liquid can alsobe a salt solution, such as a salt solution comprising cations selectedfrom the group consisting of sodium, potassium, calcium, magnesium,iron, zinc, ammonium, and mixtures thereof, for example, in an amount offrom about 0.01 M to about 5 M.

In some embodiments, a biocompatible carrier is used. The biocompatiblecarrier can comprise an aqueous-based solution, a hydro-organicsolution, an organic solution, a non-aqueous solution, or a mixturethereof. In certain embodiments, the biocompatible carrier comprises asalt solution, such as a salt solution comprising cations, selected fromthe group consisting of sodium, potassium, calcium, magnesium, iron,zinc, ammonium, and mixtures thereof, for example, in an amount of fromabout 0.01 M to about 5 M.

Methods of Management and/or Treatment

The present disclosure also relates to methods of managing and/ortreating a disease using the microspheres or pharmaceutical compositionsdisclosed herein. For example, in some embodiments, the microspheres andcompositions are suitable for managing and/or treating tumors and/orother cancers, angiogenesis-dependent diseases, non-tumorigenicangiogenesis-dependent diseases, hepatocellular diseases, or pain, suchas pain related to the presence of a tumor or other cancer. Such cancersinclude, without limitation, liver, ovarian, breast, kidney, lung,pancreatic, thyroid, prostate, uterine, skin cancer, head and necktumors, breast tumors, Kaposi's sarcoma, and superficial forms ofbladder cancer.

The microspheres and pharmaceutical compositions can be used in passiveembolization therapies and in active embolization therapies.Embolization therapies (passive and active) can include using themicrospheres to occlude or block a vessel such as a blood vessel. Activeembolization therapies also include delivery of a therapeutic agent(e.g., a drug). For example, in active embolization therapies, themicrospheres and compositions can have a dual function: mechanicalblockage or occlusion (embolization) and localized delivery of atherapeutic agent to or near the occluded site.

The microspheres can also be used as delivery systems, for example, asdelivery systems of a therapeutic agent or drug (e.g., drug-deliverysystems), with or without embolization. For example, the method ofmanagement and/or treatment may be the result of localized (or systemic)delivery of a therapeutic agent (e.g., a drug) released from themicrospheres, either alone or in combination with embolic effects of themicrospheres (active embolization). In certain embodiments, microspheresloaded with a therapeutic agent (e.g., a drug) are administered to asite-specific location other than a blood vessel (e.g., directly into atumor mass), and no vessel embolization occurs,

In certain embodiments, methods of management and/or treatment includeadministering to a mammal in need thereof a therapeutically effectiveamount of the microspheres or a pharmaceutical composition disclosedherein. The microspheres can be administered in a completely swollen orpartially swollen state. In particular embodiments, the microspheres orcompositions are suitable for administration by injection. For example,the microspheres or compositions can be injected using a needle attachedto a syringe. In other embodiments, the microspheres or compositions areadministered by a catheter. Administration can be into a blood vessel ordirectly to the site of action, for example into a tumor mass, or into acell, organ, or tissue requiring such management and/or treatment.

In some embodiments, the microspheres or compositions are loaded with atherapeutic agent (e.g., a drug) prior to administration. In otherembodiments, the microspheres or compositions are administered incombination with a drug solution, wherein the drug solution isadministered prior to, simultaneously with, or after the administrationof the microspheres. The microspheres can maintain their general shapeand position once implanted at a desired site.

Kits

The disclosure further relates to pharmaceutical packs and kitscomprising one or more containers filled with one or more of theingredients of the aforementioned compositions. For example, the kitscan comprise microspheres, and a solution comprising one or moretherapeutic agents (e.g., drugs), wherein one, two, three, or more ofthe components can be in one, two, three, or more vials. Associated withsuch containers) can be a notice in the form prescribed by agovernmental agency regulating the manufacture, use, or sale ofpharmaceuticals or biological products, reflecting approval by theagency of the manufacture, use, or sale of the product for patient(e.g., human or other mammal) administration. The reagents of any of themethods described herein can also be included as components of a kit.

In one kit format, the microspheres are present in a liquid,physiologically compatible solution in one vial. In another kit format,the microspheres can be provided in dry form in one vial and thetherapeutic agent solution and optionally a contrast agent can beprovided in a second and/or optionally a third vial. In certainembodiments, the microspheres, optionally comprising a contrast agent,are present in one vial, and the therapeutic agent is present insolution in another vial. In this form, the contents of the two vialscan be mixed together prior to or concurrently with administration. Inother embodiments, the microspheres comprising the therapeutic agent andoptionally a contrast agent are provided in dry form in one vial. Thepowder can then be suspended in a suitable liquid prior toadministration or a second vial is provided, which contains theinjectable solution and the contents of both vials are combined prior toadministration or concurrently with administration.

Finally, in another kit format the microspheres are present in one vialand a second vial contains a pharmaceutically acceptable solutionoptionally comprising a contrast agent. The microspheres in the firstvial can be pre-loaded with a therapeutic agent, or the therapeuticagent solution can optionally be present in a third vial. Themicrospheres can then be mixed together with the therapeutic agentsolution and/or pharmaceutically acceptable solution, for example, priorto or concurrently with administration.

EXAMPLES Example 1—Synthesis of Mono-OTs-β-Cyclodextrin

NaOH (0.6 g, 15 mmol, 17 eq) was added to β-cyclodextrin (1.0 g, 0.88mmol, 1 eq) that had been dissolved in water (20 mL). The resultingsolution was stirred and then cooled to 0° C. Tosyl chloride (0.34 g; 2eq) was then added to the solution. After about twelve hours, anyremaining solid tosyl chloride was removed by filtration. Then anaqueous HCl solution (1 M) was added to the filtrate to a final pH of6-7. The formed solid was recovered by filtration and recrystallizedwith water/acetonitrile (1:1) to afford mono-tosylated β-cyclodextrin.

Example 2—Synthesis of Mono-OTs-β-Cyclodextrin

β-cyclodextrin (1.0 g, 0.88 mmol, 1 eq) was mixed with pyridine (16 mL)and stirred for 15 minutes. Meanwhile, in a separate flask, tosylchloride (0.17 g, 0.88 mmol, 1 eq) and pyridine (1.2 mL) were mixed andstirred for 15 minutes. The β-cyclodextrin was then cooled toapproximately 0° C., and the tosyl chloride solution was added to thecold β-cyclodextrin mixture. After approximately 24 hours of stirring,the pyridine was removed under reduced pressure. Acetone was then addedto the mixture. The solid portion of the mixture was then collected byfiltration and washed with cold water (2×5 mL) and acetone (2×10 mL) toafford mono-tosylated β-cyclodextrin.

Example 3—Synthesis of Mono-OTs-β-Cyclodextrin

β-cyclodextrin (1.0 g, 0.88 mmol, 1 eq) was mixed with pyridine (16 mL)and stirred for 15 minutes. N,N′-dicyclohexylcarbodiimide (DCC) was thenadded (0.18 g, 0.88 mmol, 1 eq), and the mixture was stirred for anadditional 15 minutes. Meanwhile, in a separate flask, tosyl chloride(0.17 g, 0.88 mmol, 1 eq) and pyridine (1.2 mL) were mixed and stirredfor 15 minutes. The β-cyclodextrin mixture was then cooled toapproximately 0° C., and the tosyl chloride solution was added to thecold β-cyclodextrin mixture. After approximately 24 hours of stirring,water (0.35 mL) was added, and then the liquid was evaporated. Theresulting crude solid was precipitated with DMF/acetone (1:10) beforebeing recrystallized with water to afford mono-tosylated β-cyclodextrin.

Example 4—Synthesis of Mono-OTs-β-Cyclodextrin

β-cyclodextrin (1.0 g, 0.88 mmol, 1 eq) was added into water (11 mL) andthen stirred at 60° C. until fully dissolved. The solution was thencooled to 20° C., and tosylimidizole (0.35 mg) was added. The reactionwas then stirred at room temperature for 2 hours, and NaOH (0.23 g) wasadded to the mixture. The mixture was then filtered, and NH₄Cl (0.64 g)was added to the filtrate. The mixture was then concentrated and againfiltered. The resulting solid was washed with water (2×0.5 mL) andacetone (3 mL) to afford mono-tosylated β-cyclodextrin.

Example 5—Synthesis of Mono-OTs-β-Cyclodextrin

β-cyclodextrin (1.0 g, 0.88 mmol, 1 eq) was mixed with pyridine (12 mL)and stirred for 15 minutes. 4-dimethylaminopyridine (DMAP; 5 mol %) wasthen added, and the mixture was stirred for an additional 15 minutes.Meanwhile, in a separate flask, tosyl chloride (0.45 g, 2.3 mmol, 2.6eq) and pyridine (1.2 mL) were mixed and stirred for 15 minutes. Theβ-cyclodextrin mixture was then cooled to approximately 0° C., and thetosyl chloride solution was added to the cold β-cyclodextrin mixture.After approximately 24 hours of stirring at 50° C., water (0.35 mL) wasadded, and then the liquid was evaporated. The resulting crude solid wasprecipitated with 1:10 DMF/acetone before being recrystallized withwater to afford mono-tosylated β-cyclodextrin.

Example 6—Synthesis of Mono-N₃-β-Cyclodextrin

Mono-tosylated β-cyclodextrin (0.15 g) was dissolved in water (3 mL) andthen stirred for 10 minutes before adding NaN₃ (0.06 g). The mixture wasthen heated to 80° C. for 12 hours. Then acetone (20 mL) was added tothe mixture. After stirring, the mixture was filtered, and the collectedsolid was washed with water (2 mL) and acetone (10 mL) to affordmono-N₃-β-cyclodextrin.

Example 7—Synthesis of Mono-N₃-β-Cyclodextrin

Mono-tosylated β-cyclodextrin (0.65 g) was dissolved in DMF (4 mL) andthen stirred for 10 minutes before adding NaN₃ (0.16 g). The mixture wasthen heated to 75° C. for 24 hours. Then acetone (80 mL) was added tothe mixture. After stirring, the mixture was filtered, and the collectedsolid was washed with acetone (3×10 mL) to affordmono-N₃-β-cyclodextrin.

Example 8—Synthesis of Mono-NH₂-β-Cyclodextrin

Mono-tosylated β-cyclodextrin (0.1 g, 0.08 mmol) was placed in 1 mL ofaqueous NH₄OH (28%) and heated at 60° C. for four hours. The reactionwas then placed in a mixture of acetone:water (5.4:0.6) and stirred. Asolid was recovered by filtration and washed with cold ethanol to affordmono-NH₂-β-cyclodextrin.

Example 9—Synthesis of Mono-NH₂-β-Cyclodextrin

Triphenyl phosphine (PPh₃; 0.175 g, 0.67 mmol) was added to a solutionof mono-N₃-β-cyclodextrin (0.35 g, 0.3 mmol) in DMF (6 mL). The reactionwas stirred for 4 hours at 20° C. and then poured into acetone. Theresulting solid was recovered by filtration and washed with acetone toafford mono-NH₂-β-cyclodextrin.

Example 10—Synthesis of a Functionalized β-Cyclodextrin

Mono-NH₂-β-cyclodextrin (0.03 g) was placed in a saturated aqueousNaHCO₃ solution (0.4 mL). The resulting mixture was then cooled to 0°C., and acryloyl chloride (20 μL) was added. The solution was thenstirred and poured into acetone. The resulting solid was isolated byfiltration to afford the functionalized β-cyclodextrin shown below.

Example 11—Synthesis ofN-propenoyl-1-(6-deoxy-β-D-cyclodextrin)-4-aminomethyl-1,2,3 triazole

To a solution of N-(prop-2-yn-1-yl)acrylamide (0.28 g, 2.12 mmol) inDMSO (20 mL) was added mono-N₃-β-cyclodextrin (3.0 g, 2.5 mmol) andCuSO₄ pentahydrate (0.51 g, 2.12 mmol). Then sodium ascorbate (0.85 g,4.3 mmol) that had been dissolved in water (2 mL) was added dropwise tothe reaction mixture and stirred for 12 hours. The reaction was thenprecipitated in acetone (700 mL) and cooled to 4° C. The resulting solidwas recovered by filtration to obtain a brown powder. The brown powderwas then placed in aqueous NH₄OH (8%) (20 mL) and stirred. The resultingcomposition was subjected to column chromatography to obtainN-propenoyl-1-(6-deoxy-β-D-cyclodextrin)-4-aminomethyl-1,2,3 triazole.

Example 12—Synthesis of N-(2-aminoethyl)acrylamide-β-cyclodextrin)

To a solution of mono-tosylated β-cyclodextrin (0.010 g, 0.008 mmol) inDMF (2 mL) was added N-(2-aminoethyl)acrylamide (0.130 g, 1.2 mmol) andthe mixture was irradiated in a microwave oven for 30 minutes at 200 Wand 85° C. Then acetone (50 mL) was added, and the precipitate wascollected by filtration to affordN-(2-aminoethyl)acrylamide-β-cyclodextrin).

Example 13—Microsphere Synthesis

Microspheres can be prepared using a suspension polymerization processas follows: An aqueous monomer solution (solubilized in water) isprepared, which includes (1) 90 g ofN-[tris(hydroxymethyl)methyl]acrylamide) as a first monomer, (2) 30 g ofN-propenoyl-1-(6-deoxy-β-D-cyclodextrin)-4-aminomethyl-1,2,3 triazole asa cyclodextrin derivative, and (3) 10 g of N,N′-methylenebis(acrylamide)as a crosslinking agent. Water is then added to adjust the total volumeto 500 mL, and the aqueous monomer solution is warmed to 60° C. To theaqueous monomer solution is added 5 mmol of ammonium persulfate in 15 mLof water. The mixture is then briefly stirred to achieve homogeneity.

In a similar manner, the aqueous solutions of Table 1 are prepared:

TABLE 1 Cyclodextrin- containing First monomer component Crosslinkingagent N- N-propenoyl-1-(6-O- N,N′-methylenebis [tris(hydroxymethyl)hexyl-β-D- (acrylamide) methyl]acrylamide cyclodextrin)-4-aminomethyl-1,2,3 triazole N- N-propenoyl-1-(6- N,N′-methylenebis[tris(hydroxymethyl) hexylamino-6- (acrylamide) methyl]acrylamidedeoxy-β-D- cyclodextrin)-4- aminomethyl-1,2,3 triazole MethacrylamideN-propenoyl-1-(6- N,N′-methylenebis deoxy-β-D- (acrylamide)cyclodextrin)-4- aminomethyl-1,2,3 triazole N- N-propenoyl-1-(6- N′,N′-[tris(hydroxymethyl) deoxy-β-D- diallylacrylamide methyl]acrylamidecyclodextrin)-4- aminomethyl-1,2,3 triazole

A ten-liter beaker equipped with an overhead stirrer is then chargedwith four liters (L) of mineral oil and a surfactant (e.g., Arlacel 85)(1% in weight). The aqueous monomer solution is then added to the warmedoil solution (e.g., warmed to 60° C.) with vigorous stirring. 10 mL ofthe activating agent tetraethylmethylenediamine (5 mmol) is then added,while stirring, to initiate polymerization of the monomer solution. Whenpolymerization is complete, five liters of cold water are added, and themicrospheres settle in the aqueous phase. The microspheres are thenisolated by repeated washes with water to remove all the oil. Forstorage, sodium chloride is added to a final weight percent of 0.9%.

N-propenoyl-1-(6-deoxy-β-D-cyclodextrin)-4-aminomethyl-1,2,3 triazole

N-propenoyl-1-(6-O-hexyl-β-D-cyclodextrin)-4-aminomethyl-1,2,3 triazole

N-propenoyl-1-(6-hexylamino-6-deoxy-β-D-cyclodextrin)-4-aminomethyl-1,2,3triazole Example 14—Microsphere Synthesis

Microspheres are prepared using a suspension polymerization process asfollows: An aqueous monomer solution (solubilized in water) is prepared,which includes (1) 60 g of N-[tris(hydroxymethyl)methyl]acrylamide as afirst monomer, (2) 35 g of a sodium acrylate as a second monomer, (3) 30g of N-propenoyl-1-(6-deoxy-β-D-cyclodextrin)-4-aminomethyl-1,2,3triazole as a cyclodextrin-containing component, and (4) 10 g ofN,N′-methylenebis(acrylamide) crosslinking agent. Water is then added toadjust the total volume to 500 mL, and the aqueous monomer solution iswarmed to 60° C. To the aqueous monomer solution is added 5 mmol ofammonium persulfate in 15 mL of water. The mixture is then brieflystirred to achieve homogeneity.

In a similar manner, the aqueous monomer solutions of Table 2 areprepared.

TABLE 2 Cyclodextrin- containing First monomer Second monomer componentCrosslinking agent N- Sodium acrylate N-propenoyl-1-(6-N,N′-methylenebis [tris(hydroxymethyl) deoxy-β-D- (acrylamide)methyl]acrylamide cyclodextrin)-4- aminomethyl-1,2,3 triazole N- Sodiumacrylate N-propenoyl-1-(6-O- N,N′-methylenebis [tris(hydroxymethyl)hexyl-β-D- (acrylamide) methyl]acrylamide cyclodextrin)-4-aminomethyl-1,2,3 triazole N- Sodium acrylate N-propenoyl-1-(6-N,N′-methylenebis [tris(hydroxymethyl) hexylamino-6- (acrylamide)methyl]acrylamide deoxy-β-D- cyclodextrin)-4- aminomethyl-1,2,3 triazoleMethacrylamide Sodium acrylate N-propenoyl-1-(6- N,N′-methylenebisdeoxy-β-D- (acrylamide) cyclodextrin)-4- aminomethyl-1,2,3 triazole N-Sodium acrylate N-propenoyl-1-(6- N′,N′- [tris(hydroxymethyl) deoxy-β-D-diallylacrylamide methyl]acrylamide cyclodextrin)-4- aminomethyl-1,2,3triazole

A ten-liter beaker equipped with an overhead stirrer is then chargedwith four liters (L) of mineral oil and a surfactant (Arlacel 85) (1% inweight). The aqueous monomer solution is then added to the warmed oilsolution (warmed to 60° C.) with vigorous stirring. About 10 mL of anactivating agent (tetraethylmethylenediamine; 5 mmol) is then added,while stirring, to initiate polymerization of the monomer solution.Polymerization is evidenced by a mild exotherm (e.g., 3-5° C.). Whenpolymerization is complete, five liters of cold water are added, and themicrospheres settle in the aqueous phase. The microspheres are isolatedby repeated washes with water to remove all the oil. For storage, sodiumchloride is added to a final weight percent of 0.9%.

Example 15—Preparation of Drug-Loaded Microspheres

To 100 mg of dried microspheres is added 10 mL of a solution containing10 mg of cisplatin. Due to contact with the drug solution, themicrospheres swell, and the drug is chelated by the cyclodextrin of themicrospheres. The microspheres can optionally be sterilized and thenused for injection into a patient.

In a similar manner, microspheres are loaded with carboplatin,oxaliplatin, oxiplatin, satraplatin, picoplatin, nedaplatin, triplatin,lipoplatin, spiroplatin, and iproplatin, paclitaxel, and docetaxel.

Example 16—Preparation of Drug-Loaded Microspheres

To 1.5 mL of microspheres is added 7.5 mL of a cisplatin solutioncontaining 10 mg of cisplatin. Due to contact with the drug solution,the microspheres swell, and the drug is chelated by the cyclodextrin ofthe microspheres. The microspheres can optionally be sterilized and thenused for injection into a patient.

In a similar manner, microspheres are loaded with carboplatin,oxaliplatin, oxiplatin, satraplatin, picoplatin, nedaplatin, triplatin,lipoplatin, spiroplatin, and iproplatin, paclitaxel, and docetaxel.

Example 17—Microsphere Synthesis

Microspheres were prepared using a suspension polymerization process asfollows: (1) 27.13 mmol N-[tris(hydroxymethyl)methyl]acrylamide as afirst monomer, (2) 1.46 mmol ofN-propenoyl-1-(6-deoxy-β-D-cyclodextrin)-4-aminomethyl-1,2,3 triazole (aβ-cyclodextrin acrylamide derivative) as a cyclodextrin component, and(3) 3.29 mmol N,N′-methylenebis (acrylamide) crosslinking agent weredissolved at 50° C. in 45 mL of an aqueous buffer solution. The aqueousbuffer solution consisted of 30% vol. glycerin, 1 M NaCl, 0.2 M sodiumacetate, with the pH adjusted to 6.02 with diluted acetic acid. Based onpure monomers, the weight ratio ofN-[tris(hydroxymethyl)methyl]acrylamide to theN-propenoyl-1-(6-deoxy-β-D-cyclodextrin)-4-aminomethyl-1,2,3 triazolewas about 76.4 to 23.6% w/w. Based on the total molar amount of puremonomers, the cross-linking degree was about 10.4 mol %N,N′-methylenebis (acrylamide).

The solution was filtered to remove undissolved material (e.g.,impurities) and heated at 50-52° C. 104 mg of ammonium persulfateinitiator in powder form was then added to the mixture and allowed todissolve. The ratio of initiator was approximately 1.5% w/w based on thepure monomers weight.

The monomer solution with initiator was then injected into an oil phase(also referred to as a continuous phase). The oil phase consisted of 150mL hexane and 220 mg sorbitan sesquioleate as a surfactant (Arlacel 85).The oil phase was heated to 50° C. and previously deoxygenated withargon.

Polymerization was evidenced by exothermicity and local ebulition. Afterpolymerization was complete, the resulting microspheres were washed withwater. The microspheres were sieved and isolated, with fractions of themicrospheres collected at between 63-150 μm and 150-300 μm.

Example 18—Microsphere Characterization

The microspheres of Example 17 were characterized by fourier transforminfrared spectroscopy (FTIR). The FTIR spectra were collected on aThermo Scientific Nicolet iS10 FTIR Spectrometer by adding 32 scans at 4cm⁻¹ spectral resolution. Dried microspheres (as prepared in Example 17)were compared with reference microspheres made of only cross-linkedN-[tris(hydroxymethyl)methyl]acrylamide. Compared with referencemicrospheres, the microspheres of Example 17 exhibited a peak (as shownat C1) (distinguishable from the baseline) at about 1152 cm⁻¹, which isattributed to inclusion of the cyclodextrin derivative compound. FTIRspectra showing the microspheres of Example 17 (depicted as the spectrumlabeled C) in comparison to reference microspheres (depicted as thespectra labeled A and B) is shown in FIG. 1. As shown in FIG. 1, theFTIR spectrum confirmed that theN-propenoyl-1-(6-deoxy-β-D-cyclodextrin)-4-aminomethyl-1,2,3 triazole(β-cyclodextrin acrylamide derivative) polymerized with theN-[tris(hydroxymethyl)methyl]acrylamide.

The microspheres were also observed under a microscope (10×magnification) during the washing and isolation step. Images of themicrospheres are shown in FIG. 2. The microspheres were also evaluatedwith an optical system, as shown in FIGS. 3 and 4. As shown in FIGS.2-4, the microspheres were well-shaped and substantially spherical.

Example 19—Cyclodextrin Drug Loading

Loading of platinum-based drugs was tested in both α-cyclodextrins and8-cyclodextrins. In doing so, samples of oxaliplatin and cisplatin wereindividually mixed with α-cyclodextrins or β-cyclodextrins. The varioussamples were then monitored with a 400 MHz NMR equipment in water anddeuterated water at 25° C. The H¹ NMR spectra showed consistent shiftsof the cyclodextrin signals in the presence of the platinum-based drugs.These results indicate that both α-cyclodextrins and β-cyclodextrins arecapable of loading and binding platinum-based drugs, such as oxaliplatinand cisplatin. An exemplary loading scheme is represented as follows:

Example 20—Microsphere Synthesis

Microspheres (Samples A-E) were prepared using a suspensionpolymerization process as follows: (1) 30-375 g/LN-[tris(hydroxymethyl)methyl]acrylamide as a first monomer, (2) 0-280g/L N-propargyl acrylamide as a second monomer, and (3) 29-38 g/LN,N′-methylenebis (acrylamide) crosslinking agent were dissolved atabout 50° C. in aqueous buffer solutions consisting of 30-33% vol.glycerin, 0.8-0.83 M NaCl, 0.17-0.2 M sodium acetate, with the pHadjusted to 6 with diluted acetic acid.

The amounts and concentrations of materials used in the microspheres andbuffer solutions are provided in Table 3:

TABLE 3 Sample A B C D E Concentration (g/L) 400 404 400 401 343Glycerol % vol.  33%  33%  33%  33%  31% NaCl conc. 0.85M 0.85M 0.83M0.85M  0.8M Sodium Acetate conc.  0.2M  0.2M 0.17M  0.2M 0.19M pH 6 6 66 6 N,N′-methylenebis (g/L) 29.6 33.6 34.7 33.2 37.4 (acrylamide)N-[tris(hydroxymethyl) (g/L) 370.7 294.4 255.8 221.3 30.6methyl]acrylamide N-propargyl (g/L) 0 77.7 110 149.3 279.2 acrylamideCross-Linking mol % 8.3% 8.4% 8.4% 7.6% 8.3% N-[tris(hydroxymethyl) w/w% 100:0 80:20 70:30 60:40 10:90 methyl]acrylamide: N- (ratio) propargylacrylamide

The solutions were filtered to remove undissolved material (e.g.,impurities) and heated at about 55° C. Ammonium persulfate initiator inpowder form was then added to the mixtures and allowed to dissolve. Theratio of initiator was between 0.8% and 3.6% w/w based on the puremonomers weight.

The monomer solutions (60 mL) with initiator were then each poured intoan oil phase (also referred to as a continuous phase). The oil phaseconsisted of about 300 mL paraffin oil with 1-1.5 g/L sorbitansesquioleate as a surfactant (Arlacel 83). The oil phase was heated toabout 60° C. and previously deoxygenated with argon.

Polymerization was evidenced by exothermicity. After polymerization wascomplete, the resulting microspheres were washed with water. Themicrospheres were sieved and isolated, with fractions of themicrospheres collected at between 32-75 μm and 75-106 μm.

Example 21—Microsphere Characterization

The microspheres of Example 20 were characterized by optical imaging. Anillustrative image (×333 magnification) of the microspheres containing70:30 w/w ratio of N-[tris(hydroxymethyl) methyl]acrylamide:N-propargylacrylamide (Sample C) is shown in FIG. 5. As shown therein, themicrospheres were well-shaped and substantially spherical.

The microspheres were also characterized by Raman spectroscopy. In doingso, samples of the microspheres were washed with water and freeze-dried.The dry microspheres were then analyzed by Raman spectroscopy on aThermo Scientific DXR2xi Raman Imaging Microscope by adding 100 scansusing a 532 nm laser and a 50× objective. The resulting representativespectra for the various microspheres are depicted in FIG. 6. The Ramanspectra confirmed the presence of the alkyne functional group within themicrospheres (Samples B-E) via the presence of a peak or band at betweenabout 2100 cm⁻¹ and about 2150 cm⁻¹, or at about 2123 cm⁻¹,characteristic of an alkyne vibration. No such peak was present whencompared to the control group of microspheres manufactured withoutN-propargyl acrylamide (Sample A). Additionally, the height of the peakat 2123 cm⁻¹ also increased as a function of the amount of N-propargylacrylamide monomer introduced in the formulation.

Example 22—Microsphere Synthesis by Grafting Cyclodextrin

Cyclodextrin was grafted onto the microspheres manufactured inaccordance with Example 20. In particular, volumes of 10 mL of differenttypes of microspheres (microsphere Samples B-D containing 80:20, 70:30,and 60:40 w/w ratio of N-[tris(hydroxymethyl)methyl]acrylamide:N-propargyl acrylamide) were washed with water toremove any NaCl (or saline). Using a solution of CuSO₄ as a catalyst andsodium ascorbate, 6-Monoazido-6-monodeoxy-β-Cyclodextrin was reactedwith the microspheres via a click reaction to yield cyclodextrin graftedmicrospheres. The amount of 6-Monoazido-6-monodeoxy-β-Cyclodextrin usedis provided in Table 4:

TABLE 4 N-[tris(hydroxymethyl) N-propargyl methyl]acrylamide:N-Microsphere acrylamide propargyl acrylamide Dry weight dry weight (w/wratio) (g) % β-CD-N₃ (g) 80:20 (Sample B) 1.723 18.9 1.732 70:30 (SampleC) 1.710 27.5 2.508 60:40 (Sample D) 1.613 36.6 3.138

Example 23—Microsphere Characterization

The microspheres of Example 22 were characterized by optical imaging. Anillustrative image (×333 magnification) of the microspheres containing70:30 w/w ratio of N-[tris(hydroxymethyl) methyl]acrylamide:N-propargylacrylamide grafted with 6-Monoazido-6-monodeoxy-β-Cyclodextrin is shownin FIG. 7. As shown therein, the microspheres were well-shaped andsubstantially spherical.

The microspheres were also characterized by Raman spectroscopy. In doingso, samples of the microspheres were washed with water and freeze-dried.The dry microspheres were then analyzed by Raman spectroscopy on aThermo Scientific DXR2xi Raman Imaging Microscope by adding 100 scansusing a 532 nm laser and a 50× objective. The resulting representativespectrum for microspheres containing 70:30 w/w ratio ofN-[tris(hydroxymethyl) methyl]acrylamide:N-propargyl acrylamide graftedwith 6-Monoazido-6-monodeoxy-β-Cyclodextrin is depicted in FIG. 8(Sample F). FIG. 8 also provides comparison Raman spectra for themicrospheres prior to grafting (Sample C), and for ungrafted6-Monoazido-6-monodeoxy-β-Cyclodextrin (Sample G).

As shown in FIG. 8, the alkyne band at 2123 cm⁻¹ reduced between theungrafted and grafted microspheres (Sample C vs Sample F), indicatingthat at least partial grafting of the cyclodextrin had occurred via theclick reaction. Signals attributable to the cyclodextrin were alsoenriched in the microspheres after the grafting reaction.

The microspheres were also characterized by FTIR spectroscopy. In doingso, samples of the microspheres were washed with water and freeze-dried.The dry microspheres were then analyzed using a Thermo ScientificNicolet iS5 FTIR Spectrometer. The resulting representative spectrum formicrospheres containing 70:30 w/w ratio of N-[tris(hydroxymethyl)methyl]acrylamide:N-propargyl acrylamide grafted with6-Monoazido-6-monodeoxy-β-Cyclodextrin is depicted in FIG. 9 (Sample F).FIG. 9 also provides comparison FTIR spectra for the microspheres priorto grafting (Sample C), and for ungrafted6-Monoazido-6-monodeoxy-β-Cyclodextrin (Sample G).

As shown in FIG. 9, peaks between about 1140 cm⁻¹ and about 1160 cm⁻¹,or at about 1152 cm⁻¹ were only observed in Sample F (graftedmicrospheres) and Sample G (ungrafted cyclodextrin), indicating that atleast partial grafting had occurred. In contrast, no such peak ispresent in Sample C (ungrafted microspheres).

References to approximations are made throughout this disclosure, suchas by use of the term “substantially.” For each such reference, it is tobe understood that, in some embodiments, the value, feature, orcharacteristic may be specified without approximation. For example,where qualifiers such as “about” and “substantially” are used, theseterms include within their scope the qualified words in the absence oftheir qualifiers.

Any methods disclosed herein include one or more steps or actions forperforming the described method. The method steps and/or actions may beinterchanged with one another. In other words, unless a specific orderof steps or actions is required for proper operation of the embodiment,the order and/or use of specific steps and/or actions may be modified.Moreover, sub-routines or only a portion of a method described hereinmay be a separate method within the scope of this disclosure. Statedotherwise, some methods may include only a portion of the stepsdescribed in a more detailed method.

Reference throughout this specification to “an embodiment” or “theembodiment” means that a particular feature, structure, orcharacteristic described in connection with that embodiment is includedin at least one embodiment. Thus, the quoted phrases, or variationsthereof, as recited throughout this specification are not necessarilyall referring to the same embodiment.

Similarly, it should be appreciated that in the above description ofembodiments, various features are sometimes grouped together in a singleembodiment, figure, or description thereof for the purpose ofstreamlining the disclosure. This method of disclosure, however, is notto be interpreted as reflecting an intention that any claim require morefeatures than those expressly recited in that claim. Rather, as thefollowing claims reflect, inventive aspects lie in a combination offewer than all features of any single foregoing disclosed embodiment.

The claims following this written disclosure are hereby expresslyincorporated into the present written disclosure, with each claimstanding on its own as a separate embodiment. This disclosure includesall permutations of the independent claims with their dependent claims.Moreover, additional embodiments capable of derivation from theindependent and dependent claims that follow are also expresslyincorporated into the present written description.

Without further elaboration, it is believed that one skilled in the artcan use the preceding description to utilize the present disclosure toits fullest extent. The examples and embodiments disclosed herein are tobe construed as merely illustrative and exemplary and not a limitationof the scope of the present disclosure in any way. It will be apparentto those having skill in the art, and having the benefit of thisdisclosure, that changes may be made to the details of theabove-described embodiments without departing from the underlyingprinciples of the disclosure herein.

What is claimed is:
 1. Microspheres suitable for use in therapeuticembolization, comprising: a biocompatible, polymeric material comprisinga copolymer comprising: one or more acrylamide monomers; a monomercomprising an alkyne functional group, wherein the monomer comprising analkyne functional group comprises a monomer of formula I:

or formula II:

wherein X is NR¹, wherein R¹ is selected from H, CH₃, or C1-C6 alkyl,wherein R is selected from H, CH₃, or C1-C6 alkyl, and wherein n can beany integer from 1 to 20; and a cyclodextrin or a derivative thereofgrafted to the copolymer; and a therapeutic agent, wherein the copolymercomprises between about 10% and about 90% by weight of the monomers, andbetween about 10% and about 90% by weight of the cyclodextrin, whereinthe polymeric material is crosslinked and the microspheres arenon-resorbable.
 2. The microspheres of claim 1, wherein the copolymercomprises an acrylamide selected from methacrylamide,N-[tris(hydroxymethyl)methyl]acrylamide, N,N′-methylenebis(acrylamide),and derivatives and combinations thereof.
 3. The microspheres of claim1, wherein the cyclodextrin is selected from α (alpha)-cyclodextrins, β(beta)-cyclodextrins, γ (gamma)-cyclodextrins, and derivatives andcombinations thereof.
 4. The microspheres of claim 1, wherein thecyclodextrin or a derivative thereof is incorporated onto themicrosphere after polymerization of the one or more acrylamide monomersand the monomer comprising an alkyne functional group.
 5. Themicrospheres of claim 1, wherein the polymeric material comprises anacrylamide crosslinking agent.
 6. The microspheres of claim 1, whereinthe microspheres comprise a biodegradable portion and anon-biodegradable portion, wherein the biodegradable portion comprisesthe cyclodextrin or a derivative thereof, and wherein the microspheresretain a substantially spherical shape after degradation of thebiodegradable portion.
 7. The microspheres of claim 1, wherein themicrospheres are one or more of hydrophilic, swellable, and waterinsoluble.
 8. The microspheres of claim 1, wherein the microspheres havean average diameter of from about 10 μm to about 2,000 μm.
 9. Themicrospheres of claim 1, wherein the therapeutic agent comprises a drugselected from one or more of the following: cisplatin, carboplatin,oxaliplatin, oxiplatin, satraplatin, picoplatin, nedaplatin, triplatin,lipoplatin, spiroplatin, iproplatin, docetaxel and paclitaxel.
 10. Themicrospheres of claim 6, wherein the microspheres are configured todecrease the flux of blood flow across a region of the vasculature ofthe patient by 90% or more regardless of whether the biodegradableportion of the microsphere has been degraded.
 11. Microspheres suitablefor use in therapeutic embolization, comprising: a biocompatible,polymeric material comprising a copolymer comprising: one or moreacrylamide monomers; a monomer comprising an alkyne functional group,wherein the monomer comprising an alkyne functional group comprises amonomer of formula I:

or formula II:

wherein X is NR¹, wherein R¹ is selected from H, CH₃, or C1-C6 alkyl,wherein R is selected from H, CH₃, or C1-C6 alkyl, and wherein n can beany integer from 1 to 20; and a cyclodextrin or a derivative thereofgrafted to the copolymer; and a therapeutic agent, wherein the copolymercomprises between about 10% and about 90% by weight of the monomers, andbetween about 10% and about 90% by weight of the cyclodextrin, andwherein at least a portion of each microsphere is water insoluble suchthat the microsphere retains a substantially spherical shape afterplacement in a region of a patient's vasculature body, wherein thepolymeric material is crosslinked and the microspheres arenon-resorbable such that the microspheres, when disposed in the region,are configured to decrease blood flow across the region by more than 80%after degradation of the cyclodextrin or derivative thereof.
 12. Themicrospheres of claim 11, wherein the copolymer comprises an acrylamideselected from methacrylamide, N-[tris(hydroxymethyl)methyl]acrylamide,N,N′-methylenebis(acrylamide), and derivatives and combinations thereof.13. The microspheres of claim 11, wherein the cyclodextrin is selectedfrom α (alpha)-cyclodextrins, β (beta)-cyclodextrins, γ(gamma)-cyclodextrins, and derivatives and combinations thereof.
 14. Themicrospheres of claim 11, wherein the cyclodextrin or a derivativethereof is incorporated onto the microsphere after polymerization of theone or more acrylamide monomers and the monomer comprising an alkynefunctional group.
 15. The microspheres of claim 11, wherein thepolymeric material comprises an acrylamide crosslinking agent.
 16. Themicrospheres of claim 11, wherein the microspheres comprise abiodegradable portion and a non-biodegradable portion, wherein thebiodegradable portion comprises the cyclodextrin or a derivativethereof, and wherein the microspheres retain a substantially sphericalshape after degradation of the biodegradable portion.
 17. Themicrospheres of claim 11, wherein the microspheres are one or more ofhydrophilic and swellable.
 18. The microspheres of claim 11, wherein themicrospheres have an average diameter of from about 10 μm to about 2,000μm.